Inhibitory oligonucleotide and use thereof

ABSTRACT

The inhibitory oligonucleotides (ODNs) strongly block NF-κB activation induced by TLR9 agonists and TLR7 agonists. The production of proinflammatory cytokines, such as interleukin-6 and tumor necrosis factor alpha, is inhibited by the inhibitory-ODNs. Interferon production from human PBMC induced by TLR9 agonist is prevented by the inhibitory-ODNs. These ODNs can be used as a remedy for the treatment of immune-mediated disorders such as rheumatoid arthritis, systemic lupus erythematosus (SLE), sepsis, multiple organ dysfunction syndromes.

FIELD OF THE DISCLOSURE

The present invention relates to the oligonucleotides and remedies fortreating immune-mediated disorders, using the oligonucleotides. Theimmune-mediated disorder includes autoimmune disease, graft rejection,hypersensitivity, diseases associated with the over-stimulation ofhost's immune system by autoantigens, microbes and Toll-like receptor(TLR)-mediated disease.

BACKGROUND ART

The immune system protects human body from bacterial, parasitic, fungal,viral infections and from the growth of tumor cells. Immunity can beclassified as innate immunity or as adaptive immunity. Innate immuneresponses typically occur immediately upon infection for providing of anearly barrier to infectious disease whereas adaptive immune responsesoccur later with the generation of antigen-specific long term protectiveimmunity.

However, the immune response can sometimes be unwanted and causeimmune-mediated disorder. The disorder includes autoimmune disease,graft rejection, hypersensitivity, diseases associated with theover-stimulation of host's immune system by microbes and Toll-likereceptor (TLR)-mediated disease. The autoimmune diseases results from anadaptive immune response or innate immune response or both againstendogenous and/or exogenous antigens. Foreign substances, derived frombacteria, parasites, fungi or viruses, may mimic self-proteins andstimulate the immune system to launch responses to a self-cell andtissue, resulting in the diseases including but not limited to systemiclupus erythematosus (SLE) and rheumatoid arthritis. The graft rejectionis a consequence of organ or tissue transplantation caused by the immuneresponse in the transplant recipient (host) to the transplantedorgan/tissue. When a subject is transplanted with grafts includingkidney, pancreas, heart, lung, bone marrow, cornea and skin, the subjectcan launch an immune response (rejection) against the grafts.Hypersensitivity is an inappropriate immune response that hasdeleterious effects, resulting in significant tissue damage or evendeath. The hypersensitivity is divided into four types (e.g. Types I,II, III and IV. Disease associated with the over-stimulation of host'simmune system by microbes is triggered by the infection of viruses suchas flu viruses and other microbes. In the case of flu virus andGram-negative bacterial infection, an excessive immune response to theinvaders appears to be a fatal factor in patients. The response ischaracterized by the overproduction of cytokines. Studies of septicshock syndrome demonstrate that over production/aberrant production ofcytokines can lead to rapid mortality due to cytokine-mediated lethalshock (Slifka M K, et al. J Mol Med. 2000; 78(2):74-80). Septic shockfollowing gram-negative infection is a leading cause of mortality incritically ill patients. The exaggerated production of cytokines isknown to contribute to sepsis characterized by cytokine-mediated lethalshock (Espat N J, et al. J Surg Res. 1995 July; 59 (1):153-8). Multipleorgan dysfunction syndromes (MODS) are a major cause of morbidity andmortality in severe sepsis and shock. Cytokine-mediated lethal shockresulted from over-production of host cytokines is considered a mainmechanism leading to MODS (Wang H, et al. Am J Emerg Med. 2008 July; 26(6):711-5). Toll-like receptor (TLR)-mediated disease is a disordercaused by the activation of Toll like receptors (TLRs).

TLRs are a family of receptors that recognize microbe derived molecularstructures (pathogen-associated molecular patterns or PAMPs). TLRexpressing immune cells are activated upon binding of PAMPs. TLRsrecognize a range of pathogen-derived products and activated.Lipopolysaccharide (LPS) of bacteria recognized by TLR4, lipotechoicacid and diacylated lipopeptides by TLR2-TLR6 dimmer, triacylatedlipopeptides by TLR2-TLR1 dimmer, CpG containing oligonucleotide (CpGODN) synthesized or derived from either viruses or bacteria by TLR9,bacterial flagellin by TLRS, zymosan by TLR2-TLR6 dimmer, F protein fromrespiratory syncytial virus (RSV) by TLR4, viral-derived double-strandedRNA (dsRNA) and poly I:C, a synthetic analog of dsRNA by TLR3; viral DNAby TLR9, single-stranded viral RNA (VSV and flu virus) and syntheticguanosine analogs such as imidazoquinolines and imiquimod by TLR7 andTLR8 (Foo Y. Liew, et al. Nature Reviews Immunology. Vol 5, June 2005,446-458).

In recent years, TLR activation has been connected to the pathogenesisof some of diseases including sepsis, dilated cardiomyopathy, diabetes,experimental autoimmune encephalomyelitis, systemic lupus erythematosus,atherosclerosis, asthma, chronic obstructive pulmonary disease and organfailure (Foo Y. Liew, et al. Nature Review Immunology, Vol 5, 2005,446-458). Activation of TLR9 by self DNA play an important role in thedevelopment of autoimmune diseases such as psoriasis (Gilliet M, et al.Nat. Rev. Immunol. 2008, 594-606), SLE (Christensen S R, et al. Immunity2006; 25:417-28; Barrat F J, et al. J Exp Med 2005; 202:1131-9; WellmannU, et al. Proc Natl Acad Sci USA 2005; 102:9258-63) and rheumatoidarthritis (Leadbetter E A, et al. Nature 2002; 416:603-7; Boule M W, etal. J Exp Med 2004; 199:1631-40).

It has been documented that TLR9 agonist activates both innate andadaptive immune response (Arthur M. Krieg. Nature Reviews DrugDiscovery, Vol 5. June 2006, 471-484). It was documented anoligonucleotide with a nucleotide sequence of5′-cctcctcctcctcctcctcctcct-3′ prevented proliferation of humanperipheral blood mononuclear cells (PBMCs) and production of IFNs, whichinduced by TLR9 agonists (U.S. Pat. No. 8,030,289B2).

The references cited herein are not admitted to be prior art to theclaimed invention.

SUMMARY OF THE INVENTION

The present invention provides an oligonucleotide that comprises anoligonucleotide with a formula of (CCT)nCm, wherein the n is an integerfrom 6 to 16, the m is 0, 1, or 2; with the proviso that when n is 8, mis 1 or 2.

(SEQ ID NO: 1)In the first embodiment, the present invention provides an oligonucleotide with a nucleotide sequenceof 5′-cctcctcctcctcctcctcctcctc-3′. (SEQ ID NO: 2)In another embodiment, the present invention provides an oligonucleotide with a nucleotide sequenceof 5′-cctcctcctcctcctcctcctcctcc-3′. (SEQ ID NO: 3)In another embodiment, the present invention provides an oligonucleotide with a nucleotide sequenceof 5′-cctcctcctcctcctcctcctcctcct-3′. (SEQ ID NO: 4)In another embodiment, the present invention provides an oligonucleotide with a nucleotide sequenceof 5′-cctcctcctcctcctcctcctcctcctc-3′. (SEQ ID NO: 5)In another embodiment, the present invention provides an oligonucleotide with a nucleotide sequenceof 5′-cctcctcctcctcctcctcctcctcctcc-3′. (SEQ ID NO: 6)In another embodiment, the present invention provides an oligonucleotide with a nucleotide sequenceof 5′-cctcctcctcctcctcctcctcctcctcct-3′. (SEQ ID NO: 7)In another embodiment, the present invention provides an oligonucleotide with a nucleotide sequenceof 5′-cctcctcctcctcctcctcctcctcctcctc-3′. (SEQ ID NO: 8)In another embodiment, the present invention provides an oligonucleotide with a nucleotide sequenceof 5′-cctcctcctcctcctcctcctcctcctcctcc-3′. (SEQ ID NO: 9)In another embodiment, the present invention provides an oligonucleotide with a nucleotide sequenceof 5′-cctcctcctcctcctcctcctcctcctcctcct-3′. (SEQ ID NO: 10)In another embodiment, the present invention provides an oligonucleotide with a nucleotide sequenceof 5′-cctcctcctcctcctcctcctcctcctcctcctc-3′. (SEQ ID NO: 11)In another embodiment, the present invention provides an oligonucleotide with a nucleotide sequenceof 5′-cctcctcctcctcctcctcctcctcctcctcctcc-3′. (SEQ ID NO: 12)In another embodiment, the present invention provides an oligonucleotide with a nucleotide sequenceof 5′-cctcctcctcctcctcctcctcctcctcctcctcct-3′. (SEQ ID NO: 13)In another embodiment, the present invention provides an oligonucleotide with a nucleotide sequenceof 5′-cctcctcctcctcctcctcctcctcctcctcctcctcctcct-3′. (SEQ ID NO: 14)In another embodiment, the present invention provides an oligonucleotide with a nucleotide sequenceof 5′-cctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcct-3′.(SEQ ID NO: 15)In another embodiment, the present invention provides an oligonucleotide with a nucleotide sequenceof 5′-cctcctcctcctcctcct-3′. (SEQ ID NO: 16)In another embodiment, the present invention provides an oligonucleotide with a nucleotide sequenceof 5′-cctcctcctcctcctcctcct-3′.

In another embodiment, the present invention provides a remedy fortreating immune-mediated disorder using the oligonucleotides of theinvention. The immune-mediated disorder includes autoimmune disease,graft rejection, hypersensitivity, diseases associated with theover-stimulation by of host's immune system by autoantigens, microbesand Toll-like receptor (TLR)-mediated disease.

In another embodiment, the present invention provides a remedy fortreating immune-mediated disorder using the oligonucleotides of theinvention by inhibiting the TLR activation and IFNs production inducedby TLR antagonists such as DNA vims, RNA vims and the serum from SLEpatients.

In another embodiment, the present invention provides a remedy fortreating immune-mediated disorders using the oligonucleotides of theinvention by inhibiting production of proinflammatory cytokines and byrescuing a subject from cytokine-mediated lethal shock.

In another embodiment, the present invention provides a remedy fortreating immune-mediated disorders using the oligonucleotides of theinvention by inhibiting NF-κB activation induced by TLR stimulation.

In another embodiment, the present invention provides a remedy fortreating SLE, sepsis and multiple organ dysfunction syndromes in asubject using the oligonucleotides of the invention.

In another embodiment, the present invention provides methods ofregulating an immune response in an individual, comprising administeringto an individual immunostimulatory compounds in an amount sufficient toregulate an immune response in said individual Immunoregulationaccording to the methods of the invention may be practiced onindividuals including those suffering from a disorder associated with anunwanted activation of immune response.

In another embodiment, the present invention provides methods ofinhibiting a TLR9 dependent immune response in an individual, comprisingadministering to an individual immunostimulatory compounds in an amountsufficient to prevent TLR9 dependent cytokine production in saidindividual.

In another embodiment, the present invention provides methods ofinhibiting a TLR7/8 dependent immune response in an individual,comprising administering to an individual immunostimulatory compounds inan amount sufficient to prevent TLR7/8 dependent cytokine production insaid individual.

In another embodiment, the present invention provides methods ofinhibiting a NF-κB dependent immune response in an individual,comprising administering to an individual immunostimulatory compounds inan amount sufficient to prevent NF-κB dependent cytokine production insaid individual.

In another embodiment, the present invention provides a remedy fortreating immune-mediated disorder by administering the oligonucleotidesof the invention alone or with a pharmaceutically acceptable carrier toa subject through the route of enteral, parenteral and topicaladministration or inhalation.

In another embodiment, the present invention provides a compositioncomprising therapeutically effective amount of the oligonucleotides ofthe invention for the treatment of immune-mediated disorder.

In another embodiment, the present invention provides a remedy for thetreatment of immune-mediated disorder by administering theoligonucleotides of the invention alone or in combination withadditional active ingredients.

In the last embodiment, the present invention provides a remedy for thetreatment of immune-mediated disorder by administering theoligonucleotides of the invention in delivery vehicles.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-1B show suppression of NF-κB activation by inhibitory-ODNs inhuman plasmacytoid DC cell line. (FIG. 1A) The CAL-1/NFκB-GFP cell linewas designed for monitoring the activity of NF-κB transcription factorin cell-based assays. Vector encoding the GFP reporter gene driven bythe NF-κB consensus transcriptional response element was transfectedinto human plasmacytoid DC cell line; CAL-1. GFP expression was inducedby TLR9 agonist; CpG2395. (FIG. 1B) GFP expression induced by TLR9stimulation was blocked by the addition of inhibitory-ODNs.

FIGS. 2A-2D show graphs depicting the suppression ability ofinhibitory-ODNs on NF-κB activation by TLR9 stimulation in theCAL-1/NFκB-GFP cell line.

FIG. 3 shows graphs depicting the suppression ability of inhibitory-ODNson IL-6 and TNFα production from CAL-1 cells stimulated with TLR9agonist; CpG2395. Comparison of the inhibition activity between (cct)8,(cct)8c, (cct)8cc, (cct)9, (cct)10, (cct)11 and (cct)12.

FIG. 4 shows graphs depicting the suppression ability of inhibitory-ODNson IL-6 and TNFα production from CAL-1 cells stimulated with TLR9agonist; CpG2395. Comparison of the inhibition activity between (cct)8,(cct)9, (cct)10, (cct)11, (cct)12, (cct)14 and (cct)16.

FIGS. 5A-5B show graphs depicting the suppression ability ofinhibitory-ODNs on IL-6 and TNFα production from mouse DC cell line;D2SC/1. D2SC/1 cells were stimulated with TLR9 agonist; CpG1826 in thepresence of inhibitory-ODNs. (FIG. 5A) Comparison of the inhibitionactivity between (cct)8, (cct)8c, (cct)8cc and (cct)9. (FIG. 5B)Comparison of the inhibition activity between (cct)8, (cct)9, (cct)10,(cct)11, (cct)12, (cct)14 and (cct)16.

FIG. 6 shows a graph depicting the suppression activity ofinhibitory-ODNs on IFNα production from human PBMC stimulated withCpG2216. Comparison of the inhibition activity between (cct)8, (cct)9,(cct)10, (cct)11, (cct)12, (cct)14 and (cct)16 on IFNα production whichinduced by TLR9 agonist; CpG2216.

FIGS. 7A-7B show graphs depicting the suppression ability ofinhibitory-ODNs on NF-κB activation by TLR7/8 stimulation in theCAL-1/NFκB-GFP cell line. (FIG. 7A) Comparison of the inhibitionactivity between (cct)6, (cct)7 and (cct)8 for NFκB activation whichinduced by TLR7/8 agonist; Gardiquimod. (FIG. 7B) Comparison of theinhibition activity between (cct)8, (cct)9, (cct)10, (cct)11, (cct)12,(cct)14 and (cct)16 for NF-κB activation which induced by TLR7 agonist;CL264.

DETAILED DESCRIPTION OF THE INVENTION

The oligonuleotides of the present invention strongly inhibits TLR9activation. CpG containing oligonucleotides (CpG ODN) is known as a TLR9agonist [D. M. Klinman, Nat. Rev., Immunol. 4 (2004) 249-258]. Theoligonuleotides of the invention strongly inhibits the cytokinesstimulated by CpG ODN, indicating that the oligonucleotides of theinvention can be used as a remedy for the treatment of diseases relatedto TLR9 activation. Because TLR9 activation has been reported tocontribute to the development of psoriasis (Gilliet M, et al. Nat. Rev.Immunol. 2008, 594-606), SLE (Barrat F J, et al. J Exp Med 2005;202:1131-9; Wellmann U, et al. Proc Natl Acad Sci USA 2005; 102:9258-63;Christensen S R, et al. Immunity 2006; 25:417-28) and rheumatoidarthritis (Leadbetter E A, et al. Nature 2002; 416:603-7; Boule M W, etal. J Exp Med 2004; 199:1631-40), the oligonucleotides of the inventioncan be used as a remedy for the treatment of psoriasis, SLE andrheumatoid arthritis by inhibiting the TLR9 activation.

The oligonuleotides of the present invention strongly inhibits IFNproduction from human PBMC induced by TLR9 agonist. Because the elevatedproduction of IFNs has been reported to contribute to the development ofSLE (Barrat F J, et al. J Exp Med 2005; 202:1131-9; Wellmann U, et al.Proc Natl Acad Sci USA 2005; 102:9258-63), the oligonucleotides of theinvention can be used as a remedy for the treatment of SLE by inhibitingIFN production.

The oligonuleotides of the present invention strongly inhibits thecytokines production which induced by TLR7/8 agonist. Theoligonucleotides of the present invention can be used as a remedy forthe treatment of Toll-like receptor (TLR)-mediated disease by inhibitingTLR7 or TLR8.

It has been demonstrated that injection of TLR9 agonist; CpG ODN withthe D-galactosamine (D-Gal) into mice induced hyper immune reactions.The model mice died within 12 to 24 h. Analyses of plasma cytokinesrevealed over-production of proinflammatory cytokines such as TNFα(Marshall A J, et al. Infect Immun 1998 April; 66(4):1325-33; Peter M,Bode K, et al. Immunology. 2008 January; 123(1):118-28). Theoligonucleotides of the present invention strongly inhibits theproduction of TNFα from mouse cells induced by TLR9 stimulation Becausethe cytokine-mediated lethal shock contributes to the septic shock(Slifka M K, et al. J Mol Med. 2000; 78(2):74-80; Espat N J, et al. JSurg Res. 1995 July; 59(1):153-8) and multiple organ dysfunctionsyndromes (MODS) (Wang H, et al. Am J Emerg Med. 2008 July;26(6):711-5), the oligonucleotides of the present invention can be usedas a remedy for the treatment of sepsis and MOGS by rescuing the hostfrom cytokine-mediated lethal shock.

NF-κB is clearly one of the most important regulators of proinflammatorygene expression. Activation of the NF-κB plays a central role ininflammation through its ability to induce transcription ofproinflammatory cytokines (Baldwin (Jr) A S, et al. Annu Rev Immunol.1996, 649-683). It has been demonstrated that NF-κB plays a role inconstitutive IL-6 production in rheumatoid arthritis (RA) synovialfibroblasts (Miyazawa K, et al. Am J Pathol 1998, 793-803). NF-κB isintimately involved in activation of inflammatory genes by IL-1 or TNFαin human monocytes (Schottelius A J, et al. J Biol Chem 1999,31868-31874). The number of NF-κB positive cells correlates with thedegree of gastritis. Similarly, there is evidence of NF-κB activation ininflammatory bowel disease, where lamina propria macrophages displayactivated NF-κB (Neurath M F, et al. Ann NY Acad Sci 1998, 859:149-159).

The activation of TLRs by the ligands induces the activation oftranscription factors such as NF-κB and interferon responsive factors(IRFs). Those activated transcription factors further induce theproduction of cytokines such as interleukin-6 (IL-6), tumor necrosisfactor alpha (TNFα) and the interferons (IFNs).

The oligonuleotides of the invention strongly inhibits NF-κB activationinduced by TLR stimulation, indicating that the oligonucleotides of theinvention can be used as a remedy for the treatment of diseases relatedto NF-κB activation. As NF-κB activation has been reported to contributeto the development of rheumatoid arthritis, gastritis and inflammatorybowel disease, the oligonucleotides of the invention can be used as aremedy for the treatment of rheumatoid arthritis, gastritis andinflammatory bowel disease by inhibiting the NF-κB activation.

Unless otherwise noted, all terms in the invention have the same meaningas commonly understood by one of ordinary skill in the art to which thisdisclosure belongs. The singular terms “a,” “an,” and “the” includeplural referents unless context indicates otherwise. Similarly, the word“or” is intended to include “and” unless the context indicatesotherwise. In case of conflict, the present specification, includingexplanations of terms, will control. In addition, the materials, methodsand examples are illustrative only and not intended to be limiting.Treat, treating or treatment shall have the same meaning withoutconcerning the grammar. Similarly, prevent, preventing or preventionshall have the same meaning without concerning the grammar.“Oligonucleotide”: An oligonucleotide means multiple nucleotides (i.e.molecules comprising a sugar (e.g. deoxyribose) linked to a phosphategroup and to an exchangeable organic base, which is either a substitutedpyrimidine (Py) (e.g., cytosine (C), thymine (T)) or a substitutedpurine (Pu) (e.g., adenine (A) or guanine (G)). The term oligonucleotideas used herein refers to oligodeoxyribonucleotide (ODN). Theoligonucleotide can be obtained from existing nucleic acid sources(e.g., genomic or cDNA), but are preferably synthetic. Theoligonucleotide of the invention can be synthesized by a variety ofautomated nucleic acid synthesizers available in the market. Theseoligonucleotides are referred to as synthetic oligonucleotides.

“Chemical modification”: The oligonucleotide disclosed in the inventioncan encompass various chemical modifications, in comparison to naturalDNA, involving a phosphodiester internucleoside bridge, a ribose unitand/or a natural nucleoside base (adenine, guanine, cytosine, thymine).The modifications can occur either during or after synthesis of theoligonucleotide. During the synthesis, modified bases can beincorporated internally or on its end. After the synthesis, themodification can be carried out using the active groups (via an aminomodifier, via the 3′ or 5′ hydroxyl groups, or via the phosphate group).The skilled person knows examples of chemical modifications. Anoligonucleotide according to the invention may have one or moremodifications, wherein each modification is located at a particularphosphodiester internucleoside bridge and/or at a particular ribose unitand/or at a particular natural nucleoside base position in comparison toan oligonucleotide of the same sequence, which is composed of naturalDNA. The chemical modification includes “back bone modification” of theoligonucleotide of the invention. As used herein, the modified back boneof the oligonucleotide of the invention includes, but not limited to the“phosphorothioate backbone” that refers to a stabilized sugar phosphatebackbone of a nucleic acid molecule in which a non-bridging phosphateoxygen is replaced by sulfur at least one internucleotide linkage. Inone embodiment a non-bridging phosphate oxygen is replaced by sulfur ateach and every internucleotide linkage Other back bone modificationsdenote the modification with nonionic DNA analogs, such as alkyl- andaryl-phophonates (in which the charged phosphonate oxygen is replaced byan alkyl or aryl group), phophodiester and alkylphosphotriesters, inwhich the charged oxygen moiety is alkylated. In other examples, theoligonucleotide can be is a phosphorothioate/phosphodiester chimera. Thechemical modification also includes the base substitutions of theoligonucleotide disclosed in the invention. The substituted purines andpyrimidines can be C-5 propyne pyrimidine and 7-deaza-7-substitutedpurine. The substituted purines and pyrimidines include but are notlimited to adenine, cytosine, guanine, and thymine, and other naturallyand non-naturally occurring nucleobases. The chemical modification ofthe oligonucleotide of the invention further includes the modificationof the bases of the oligonucleotide. A modified base is any base whichis chemically distinct from the naturally occurring bases typicallyfound in DNA such as T, C, G and A, but which share basic chemicalstructures with these naturally occurring bases. The oligonucleotide ofthe invention can be modified by using cytidine derivatives. The term“cytidine derivative” refers to a cytidine-like nucleotide (excludingcytidine) and the term “thymidine derivative” refers to a thymidine-likenucleotide (excluding thymidine). In addition, the oligonucleotides ofthe invention can be chemically modified by linking a diol, such astetraethyleneglycol or hexaethyleneglycol, at either or both termini ofthe oligonucleotide.

“Immune-mediated disorder”: An immune-mediated disorder is a diseasecaused by an unwanted immune response in a subject. The disorderincludes autoimmune disease, graft rejection, hypersensitivity, diseasesassociated with the over-stimulation of host's immune system by microbesand diseases associated with TLR activation. The oligonucleotidedisclosed in the invention can be used as a remedy to treat theimmune-mediated disorder.

“Immune response”: A response of a cells of the immune system, such as aB cell, T cell, natural killer cell, dendritic cell, neutrophil andmacrophage to a stimulus. The response includes innate immune responseand adaptive (specific or acquired) immune response. The adaptive(specific or acquired) immune response includes humoral immune responseand cellular immune response.

“Prevent or treat immune-mediated disorder”: As used herein, preventrefers to prevent the full development of an immune-mediated disorder ina subject; treat refer a therapeutic intervene in a subject so as toameliorate a sign or symptom of, halt the progression of, or eliminatepathological condition of the immune-mediated disorder.

“Subject”: As used herein, a subject refers to a human or non-humanvertebrate. Non-human vertebrates are non-human primates, livestockanimals and companion animals. The oligonucleotide of the invention canbe administered to prevent or/and treat immune-mediated disorder in asubject.

“Autoimmune diseases”: The term “autoimmune disease” refers to a diseasecaused by a breakdown of self-tolerance such that the adaptive andinnate immune system responds to self antigens and mediates cell andtissue damage Autoimmune diseases are frequently characterized by meansof their involvement of single organ or single cell-types or involvementof multiple organs or tissue systems. Autoimmune diseases have also beenreferred to as “collagen,” or “collagen-vascular” or “connective tissue”diseases. Autoimmune disorders are frequently associated withhypersensitivity reactions. The oligonucleotides of the invention can beuseful for treating and/or preventing various types of autoimmunediseases. Specific, non-limiting examples of autoimmune disorders aresystemic lupus erythematosus, insulin-dependent (type I) diabetesmellitus, inflammatory arthritis, rheumatoid arthritis, multiplesclerosis, autoimmune hepatitis, chronic aggressive hepatitis,autoimmune hemolytic anemia, autoimmune thrombocytopenia, autoimmuneatrophic gastritis of pernicious anemia, autoimmune encephalomyelitis,autoimmune orchitis, acquired hemophilia, ankylosing spondylitis,antiphospholipid syndrome, Beh.cedilla et's syndrome, cardiomyopathy,chronic inflammatory demyelinating polyneuropathy, cicatricialpemphigoid, cold agglutinin disease, polymyositisdermatomyositis,discoid lupus, sympathetic ophthalmia, essential mixed cryoglobulinemia,fibromyalgia, fibromyositis, Guillain-Barr syndrome, idiopathicpulmonary fibrosis, idiopathic thrombocytopenic purpura, IgAnephropathy, juvenile arthritis, systemic sclerosis, polyarteritisnodosa, polychondritis, dermatomyositis, primary agammaglobulinemia,primary biliary cirrhosis, hyperimmunoglobulin E, progressive systemicsclerosis, psoriasis, Reiter's syndrome, sarcoidosis, stiff-mansyndrome, uveitis, vasculitis, vitiligo, Hashimoto's thyroiditis,Goopasture's disease, pernicious anemia, Addison's disease,dermatomyositis, Sjogren's syndrome, dermatomyositis, myasthenia gravis,Grave's disease, uveitis, allergic encephalomyelitis,glomerulonephritis, and the like (N Engl J Med, Vol. 345, No. 5, Aug. 2,2001, p 340-350). DNA or RNA released from DNA- or RNA-containingmicrobes could stimulate the production of autoantibody specific to selfRNA- or DNA-containing complexes and consequently led to an autoimmunedisease, including but not limited to SLE.

“Hypersensitivity”: A hypersensitivity is referred to the disorderswherein tissue injury occurs as a result of a humoral or cell-mediatedresponse to antigens of endogenous or exogenous origin and has beenclassified into four types. Type I hypersensitivity (frequently referredto as anaphylactic, immediate-type, atopic, reagenic, IgE-mediatedhypersensitivity reactions or allergy) generally result from the releaseof pharmacologically active substances such as histamine, slow-reactingsubstance of anaphylaxis (SRS-A), and eosinophilic chemotactic factor(ECF) form IgE-sensitized basophils and mast cells after contact with aspecific exogenous antigen. Type I hypersensitivity includes, but notlimited to, allergic extrinsic asthma, seasonal allergic rhinitis andsystemic anaphylaxis. Type II hypersensitivity (also referred to ascytotoxic, cytolytic complement-dependent or cell-stimulatinghypersensitivity reaction) results when antibody reacts with antigeniccomponents of cells or tissue elements or with an antigen or hapten,which has become intimately coupled to cells or tissue. Type IIhypersensitivity includes, but not limited to, autoimmune hemolyticanemia, erythroblastosis fetalis and Goodpasture's disease. Type IIIhypersensitivity (also referred to as toxic complex, soluble complex, orimmune complex hypersensitivity reactions) results from the depositionof soluble circulating antigen-antibody complexes in vessels or intissues, with accompanying acute inflammatory reactions at the site ofimmune complex deposition. Type III hypersensitivity includes, but notlimited to, Arthurs reaction, serum sickness, systemic lupuserythematosis, and certain types of glomerulonephritis. Type IVhypersensitivivity (frequently called cellular, cell-mediated, delayed,or tuberculin-type hypersensitivity reactions) are caused by sensitizedT-lymphocytes which result from contact with a specific antigen. Type IVhypersensitivity includes, but not limited to, contact dermatitis andallograft rejection (Richard A. et al Immunology, Fifth Edition, 2003,W.H. FREEMAN AND COMPANY).

“Diseases associated with the over-stimulation of host's immune systemby microbes”: Microbe invasion, if severe, sometimes can cause systemicinflammatory response in a subject, leading to diseases associated withthe over-stimulation of host's immune system by microbes. The events inthe development of the diseases, such as in the case of influenza A(H5N1) or bacterial infection, include the significantly elevated bloodlevels of TNFα, interleukin-1 (IL-1), IL-6, IL-12, interferon alpha(IFN-α), interferon beta (IFN-β), interferon gamma (IFN-γ), chemokinesinterferon-inducible protein 10, monocyte chemoattractant protein 1,interleukin-8, interleukin-1β, and monocyte chemoattractant protein 1.Such responses can result in cytokine-mediated lethal shock that isresponsible in part for the sepsis, ARDS, and multiorgan failureobserved in many patients (The Writing Committee of the World HealthOrganization (WHO) Consultation on Human Influenza A/H5. Avian InfluenzaA (H5N1) Infection in Humans N Engl J Med 2005; 353:1374-85). Thesignificantly elevated blood level of cytokines followed microbeinfection is termed by hypercytokinemia (hypercytokinaemia) or acytokine storm. The research suggested that patients who contract birdflu or SARS may need drugs that suppress the immune response in additionto anti-viral drugs. The oligonucleotide of the invention can be used totreat and/or prevent the diseases associated with the stimulation ofhost's immune system by microbes in a subject. The microbes causing thediseases includes, but not limited to, viruses, bacteria, fungi,parasites and etiological agents of Spongiform encephalopathies. Thevirus that cause the diseases associated with the over-stimulation ofhost's immune system by microbes include: SARS CoV, influenza viruses,avian flu virus HIV-1, polio viruses, hepatitis A virus; enteroviruses,human Coxsackie's viruses, rhinoviruses, echoviruses, equineencephalitis viruses, rubella viruses, dengue viruses, encephalitisviruses, yellow fever viruses, corona viruses, vesicular stomatitisviruses, rabies viruses, Ebola viruses, parainfluenza viruses, mumpsvirus, measles virus, respiratory syncytial virus, influenza viruses,Hantan viruses, bunga viruses, phlebovimses, Nairo viruses, hemorrhagicfever viruses; reovimses, orbiviurses and rotaviruses, Hepatitis Bvirus, parvoviruses, papilloma viruses, polyoma viruses, adenoviruses,herpes simplex virus (HSV) 1 and HSV-2, varicella zoster virus,cytomegalovirus (CMV), herpes viruses, variola viruses, vacciniaviruses, pox viruses, African swine fever virus, the etiological agentsof Spongiform encephalopathies, delta hepatitis virus, Hepatitis Cvirus, foot and mouth disease virus and avian flu virus. The bacteriathat can cause the diseases associated with the over-stimulation ofhost's immune system by microbes include: Helicobacter pyloris, Boreliaburgdorferi, Legionella pneumophilia, Mycobacteria sps (such as. M.tuberculosis, M. avium, M. E intracellulare, M. kansaii, M. gordonae),Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis,Listeria monocytogenes, Group A Streptococcus, Group B Streptococcus,Streptococcus, Streptococcus faecalis, Streptococcus bovis,Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenicCarnpylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillusantracis, corynebacterium diphtheriae, corynebacterium sp.,Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridiumtetani, Enterobacter aerogeytes, Klebsiella pneumoniae, Pasteurellamultocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillusmoniliformis, Treponema palladium, Treponema pertenue, Leptospira, andActinomyces israelli. The fungi that can cause the diseases associatedwith the over-stimulation of host's immune system by microbes include,but not limited to, Cryptococcus neoformans, Histoplasma capsulatum,Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis,Candida albicans. The parasites that can cause the diseases associatedwith the over-stimulation of host's immune system by microbes include:Plasmodium falciparum and Toxoplasma gondii.

“Graft rejection”: The graft rejection is an immune-mediated disordercaused by organ or tissue transplantation, Transplantation means thetransfer of transplants (grafts) from a donor to a recipient. Grafts arethe living cells, tissues, or organs transplanted from a donor to arecipient. An autograft is the a graft transferred of one's own tissuefrom one location to another; a syngeneic graft (isograft) is a graftbetween identical twins; an allogeneic graft (homograft) is a graftbetween genetically dissimilar members of the same species; and axenogeneic graft (heterograft) is a transplant between members ofdifferent species. When a subject is the recipient of an allogeneicgraft or a xenogeneic graft, the body can produce an immune responseagainst the donor tissue. In this situation, there is a clear need tosuppress the immune response, in order to avoid rejection of the graft(Richard A. et al. Immunology, Fifth Edition, 2003, W.H. FREEMAN ANDCOMPANY). The oligonucleotides of the present invention are useful whenadministered for the prevention of the graft rejection. Examples of thegrafts are heart, kidney, liver, medulla ossium, skin, cornea, lung,pancreas, intestinum tenue, limb, muscle, nervus, duodenum, small-bowel,pancreatic-islet-cell, and the like. In some case, the recipient may bean animals as defined in “subject” of the invention.

“Toll-like receptor (TLR)-mediated diseases”: A Toll-like receptor(TLR)-mediated disease means an immune mediated disorder related to theactivation of members of the TLR family. The disease includes, but notlimited to, the diseases include but not limited to, sepsis associatedwith the activation of TLR4 by lipopolysaccharide (LPS), dilatedcardiomyopathy associated with the activation of TLR2, 3, 4, 9, diabetesassociated with the activation of TLR2,3,4,9, experimental autoimmuneencephalomyelitis associated with the activation of TLR3, systemic lupuserythematosus associated with the activation of TLR9, atherosclerosisassociated with the activation of TLR4, asthma associated with theactivation of TLR4 by LPS, chronic obstructive pulmonary diseaseassociated with the activation of TLR4, EAE associated with theactivation of TLR4 and organ failure associated with the activation ofTLR4 (Foo Y. et al. Nature Review Immunology, Vol 5, 2005, 446-458).CpG-containing DNA (a TLR9 agonist) derived from a nucleicacid-containing infectious agent could be identified from SLE serum thatinduces an efficient immune response dominated by IFN-α secretion thatis thought to contribute the development of SLE. The oligonucleotides ofthe present invention can be administered for treating and/or preventthe Toll-like receptor (TLR)-mediated diseases including but not limitedto SLE in a subject.

“CpG ODN”: It has been documented that TLR9 agonist activates bothinnate and adaptive immune response (Arthur M. Krieg. Nature ReviewsDrug Discovery, Vol 5. June 2006, 471-484). CpG containingoligonucleotides (CpG ODN) is a TLR9 agonist [D. M. Klinman, Nat. Rev.,Immunol 4 (2004) 249-258]. Based on the functional characteristics, CpGODNs are divided into three types (Tomoki Ito, et al. Blood, 2006, Vol107, Num 6: 2423-2431). A-type CpG ODN activates human plasmacytoiddendritic cells (pDCs) to produce large amount of type I interferon(IFN-a/β) and strongly activates natural killer cells (NK cells). B-typeCpG ODN primarily activates B cells, resulting in their proliferationand antibody secretion. C-type CpG ODN shares the activities of both A-and B-type CpG ODN. As a TLR9 agonist, CpG ODN such as CpG 2216 or CpG2006 or CpG 2395 can be endocytosed into a cellular compartment wherethey are exposed to and activate TLR9. In pDC, TLR9 activation initiatea rapid innate immune response that is characterized by the secretion ofpro-inflammatory cytokines [IL-6, tumor-necrosis factor-α (TNFα)], thesecretion of type I interferon (IFN) and the secretion of secretion ofIFN-inducible chemokines. Through both IFN-dependent and IFN-independentpathways, innate immune cells including natural killer (NK) cells,monocytes and neutrophils are secondarily activated by the pDC. B cellsactivated through TLR9 have a greatly increased sensitivity to antigenstimulation and efficiently differentiate into antibody-secreting cells,and therefore contributing to the adaptive immune response, especiallyhumoral immune response. pDC activated through TLR9 secrete IFNα, whichdrives the migration and clustering of pDC to lymph nodes and othersecondary lymphoid tissues where the pDC activates naive and memory Tcells, assists the cross-presentation of soluble protein antigens toCD8+ cytotoxic T lymphocyte (CTL) and promotes strong TH1 biasedcellular CD4 and CD8 T-cell responses. Based on the above mentionedfindings, it is obvious that the agents that antagonize the activity ofCpG ODN can be used to treat or prevent the immune-mediated disorder byinhibiting both innate and adaptive immune response.

“pharmaceutically acceptable carrier”: A pharmaceutically acceptablecarrier denotes one or more solid or liquid filler, diluents orencapsulating substances that are suitable for administering theoligonucleotide of the invention to a subject. The carrier can beorganic, inorganic, natural or synthetic. The carrier includes any andall solutions, diluents, solvents, dispersion media, liposome,emulsions, coatings, antibacterial and anti-fungal agents, isotonic andabsorption delaying agents, and any other carrier suitable foradministering the oligonucleotide of the invention and their use is wellknown in the art. The pharmaceutically acceptable carriers are selecteddepending on the particular mode of administration of theoligonucleotide. The parenteral formulations usually comprise injectablefluids that include pharmaceutically and physiologically acceptablefluids such as water, physiological saline, balanced salt solutions,aqueous dextrose, glycerol or the like as a vehicle. For solidcompositions (e. g., powder, pill, tablet, or capsule forms),conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically-neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

“therapeutically effective amount”: In order to treat or prevent animmune-mediated disorder, a therapeutically effective amount of anoligonucleotide of the invention is administered to a subject. The“therapeutically effective amount” of one of the oligonucleotides meansa sufficient amount of the oligonucleotide used to achieve a desiredresult of treating or preventing an immune-mediated disorder in asubject. The oligonucleotides of the present invention may be employedin pure form or in pharmaceutically acceptable carriers. Alternatively,the oligonucleotides may be administered as pharmaceutical compositions.The “amount” in the invention shall refer to a dose. The dose can bedetermined by standard techniques well known to those skilled in the artand can vary depending the factors including, but not limited to thesize or/and overall health of the subject or the severity of thedisease. Introduction of the oligonucleotide of the invention can becarried out as a single treatment or over a series of treatments.Subject doses of the oligonucleotide of the invention for theadministration range from about 1 μg to 100 mg per administration.However, doses for the treatment of immune-mediated disorder may be usedin a range of 10 to 1,000 times higher than the doses described above.The more preferable doses can be adjusted to provide the optimumtherapeutic effect by those skilled in the art, for example, by theattending physician within the scope of sound medical judgment.

“Route of administration”: For clinical use, the oligonucleotides of theinvention can be administered alone or formulated in a pharmaceuticalcomposition via any suitable route of administration that is effectiveto achieve the desired therapeutic result. The “route” of administeringthe oligonucleotide of the invention shall mean the enteral, parenteraland topical administration or inhalation. The enteral routes ofadministration of the oligonucleotide of the invention include oral,gastric, intestinal, and rectal. The parenteral route includesintravenous, intraperitoneal, intramuscular, intrathecal, subcutaneous,local injection, vaginal, topical, nasal, mucosal, and pulmonaryadministration. The topical route of administration of theoligonucleotide of the invention denotes the application of theoligonucleotide externally to the epidermis, to the buccal cavity andinto the ear, eye and nose.

“pharmaceutical composition” A pharmaceutical composition shall mean thecomposition comprising an therapeutically effective amount of theoligonucleotide of the invention with or without a pharmaceuticallyacceptable carrier. The pharmaceutical compositions can comprise one ormore oligonucleotides of the invention. The composition includes but notlimited to aqueous or saline solutions, particles, aerosols, pellets,granules, powders, tablets, coated tablets, (micro) capsules,suppositories, syrups, emulsions, suspensions, creams, drops and otherpharmaceutical compositions suitable for use in a variety of drugdelivery systems. The compositions may be administered parenterally,orally, rectally, intravaginally, intraperitoneally, topically (in adosage form as powders, ointments, gels, drops or transdermal patch),bucally, or as an oral or nasal spray. In all cases, the compositionmust be sterile and stable under the conditions of manufacture andstorage and preserved against the microbial contamination.Pharmaceutical compositions of this invention for parenteral injectioncomprise pharmaceutically-acceptable sterile aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions, as well as sterilepowders for reconstitution into sterile injectable solutions ordispersions just prior to use. The oligonucleotide of the invention canbe suspended in an aqueous carrier, for example, in an isotonic buffersolution at a pH of about 3.0 to about 8.0, preferably at a pH of about3.5 to about 7.4, 3.5 to 6.0, or 3.5 to about 5.0. The buffer solutionincludes sodium citrate-citric acid and sodium phosphate-phosphoricacid, and sodium acetate-acetic acid buffers. For oral administration,the composition will be formulated with edible carriers to form powderstablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions and the like. For solid compositions, conventional non-toxicsolid carriers can include pharmaceutical grades of mannitol, lactose,starch, or magnesium stearate. For buccal administration, thecomposition will be tablets or lozenges in conventional manner. Forinhalation, the composition will be an aerosol spray from pressurizedpacks or a nebulizer or a dry powder and can be selected by one of skillin the art. In some cases, in order to prolong the effect of theoligonucleotide of the invention, the oligonucleotides of the inventionare also suitably administered by sustained-release systems. Theoligonucleotide of the invention can be used in a liquid suspension ofcrystalline or amorphous material with poor water solubility to slow thereleasing of the oligonucleotide. Alternatively, delayed releasing of aparenterally administered drug form of the oligonucleotide isaccomplished by dissolving or suspending the oligonucleotide inhydrophobic materials (such as an acceptable oil vehicle). Injectabledepot forms are made by entrapping the oligonucleotide in liposomes ormicroemulsions or other biodegradable semi-permeable polymer matricessuch as polylactide-polyglycolide, poly (orthoesters) and poly(anhydrides).

“Active ingredients”. The oligonucleotides of the invention can be usedalone, in combination with themselves, in a pharmaceutically acceptablecarrier, in combination with one or more additional active ingredients.The administration of the oligonucleotide of the invention andadditional active ingredients can be sequential or simultaneous. Theactive ingredients include non-steroidal anti-inflammatory agents,steroids, nonspecific immunosuppressive agent, biological responsemodifier, chemical compound, small molecule, nucleic acid molecule andTLR antagonists. The active ingredients also denote the agents thatsuppress the immune activation by antagonizing chemochines, by inducingthe generation of regulatory T cells (CD4+CD25+ T cells), by inhibitinga complement, matrix metalloproteases and nitric oxide synthase, byblocking costimulatory factors and by inhibiting the signaling cascadesin the immune cells. The non-steroidal anti-inflammatory agents include,but unlimited to, diclofenac, diflunisal, etodolac, flurbiprofen,ibuprofen, indomethacin, ketoprofen, ketorolac, nabumetone, naproxen,oxaprozin, piroxicam, sulindac, tohnetin, celecoxib and rofecoxib. Thesteroids include, but unlimited to, cortisone, dexamethasone,hydrocortisone, methylprednisolone, prednisolone, prednisone, andtriamcinolone. A nonspecific immunosuppressive agent means the agentused to prevent the development of immune-mediated disorder. Thenonspecific immunosuppressive agents include but not limited tocyclophosphamide, cyclosporine, methotrexate, steroids, FK506,tacrolimus, mycophenolic acid and sirolimus. The biological responsemodifier includes a recombinant interleukin-1-receptor antagonist(Kineret or anakima), a soluble p75 TNFα receptor-IgG1 fusion protein(etanercept or Enbrel), or a monoclonal antibody against TNFα(infliximab or RemicadeX). The agents also include Interferon beta-la,interleukin-10 and TGFβ.

“delivery vehicle”: The oligonucleotides of the invention can beadministered in/with a delivery vehicle or in a form linked with avehicle. The vehicle includes, but not limited to, sterol (e.g.,cholesterol), cochleates, emulsomes, ISCOMs; a lipid (e.g., a cationiclipid, anionic lipid), liposomes; ethylene glycol (PEG); live bacterialvectors (e.g., Salmonella, Escherichia coli, bacillus Calmette-Gurin,Shigella, Lactobacillus), live viral vectors (e.g., Vaccinia,adenovirus, Herpes simplex), virosomes, virus-like particles,microspheres, nucleic acid vaccines, polymers (e.g.,carboxymethylcellulose, chitosan), polymer rings and a targeting agentthat recognizes target cell by specific receptors.

“Pegylation”: Pegylation is the process of covalent attachment of poly(ethylene glycol) polymer chains to another molecule, normally a drug ortherapeutic protein. Pegylation is routinely achieved by incubation of areactive derivative of PEG with the target agent. The pegylated agentcan “mask” the agent from the host's immune systems, increase thehydrodynamic size of the agent which prolongs its circulatory time. Theoligonucleotides of the invention can be pegylated.

EXAMPLES

The invention will now be described in more detail in the followingExamples. But the invention is not limited to these Examples. In theseExamples, herein, experiments using commercially available kits andreagents were done according to attached protocols, unless otherwisestated. The skilled artisan will appreciate that the oligonucleotides ofthe present invention can easily be applied to treat an immune-mediateddisorder. The present invention will now be demonstrated by thefollowing non-limiting examples.

The all oligonucleotides (ODNs) used in the example were synthesized inHokkaido System Science Co. Ltd (Sapporo, Japan). TLR9 stimulatory ODNswere CpG2395 (5′-tcgtcgttttcggcgcgcgccg-3′, SEQ ID No: 17), CpG1826(5′-tccatgacgttcctgacgtt-3′, SEQ ID No: 18), CpG2216(5′-gggggacgatcgtcgggggg-3′, SEQ ID No: 19). Other ODNs used in theexamples were (CCT)6 (5′-cctcctcctcctcctcct-3′, SEQ ID No: 15), (CCT)7(5′-cctcctcctcctcctcctcct-3′, SEQ ID No: 16), (CCT)8(5′-cctcctcctcctcctcctcctcct-3′, SEQ ID No: 20), (CCT)8C(5′-cctcctcctcctcctcctcctcctc-3′, SEQ ID No: 1), (CCT)8CC(5′-cctcctcctcctcctcctcctcctcc-3′, SEQ ID No: 2), (CCT)9(5′-cctcctcctcctcctcctcctcctcct-3′, SEQ ID No: 3), (CCT)10(5′-cctcctcctcctcctcctcctcctcctcct-3′ SEQ ID No: 6), (CCT)10C(5′-cctcctcctcctcctcctcctcctcctcctc-3 SEQ ID No: 7), (CCT)10CC(5′-cctcctcctcctcctcctcctcctcctcctcc-3 SEQ ID No: 8), (CCT)11(5′-cctcctcctcctcctcctcctcctcctcctcct-3′, SEQ ID No: 9), (CCT)11C(5′-cctcctcctcctcctcctcctcctcctcctcctc-3′, SEQ ID No: 10), (CCT)11CC(5′-cctcctcctcctcctcctcctcctcctcctcctcc-3′, SEQ ID No: 11), (CCT)12(5′-cctcctcctcctcctcctcctcctcctcctcctcct-3′ SEQ ID No: 12), (CCT)14(5′-cctcctcctcctcctcctcctcctcctcctcctcctcctcct-3′, SEQ ID No: 13) and(CCT)16 (5′-cctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcct-3′, SEQ IDNo: 14). All reagents used to manipulate the oligonucleotides (ODNs) inthe following examples were pyrogen-free.

Example 1

Effect of Inhibitory-ODNs on NF-κB Activation Induced by TLR9Stimulation

<Experimental Method>

CAL-1/NFκB-GFP cell line was established for monitoring the activity ofNF-κB transcription factor in cell-based assays. Vector encoding the GFPreporter gene driven by the NFκB consensus transcriptional responseelement was transfected into human plasmacytoid DC cell line; CAL-1 byelectroporation. Transfected cells were further selected with zeocin.(FIG. 1A) GFP expression induced by TLR9 agonist; CpG2395 was evaluated.Briefly, CAL-1/NFκB-GFP cells (1×10⁵/well) were plated in 96-wellflat-bottomed plate (Costar) and cultured with or without CpG2395 (1μM). The cells were incubated at 37° C. in a 5% CO₂ humidified incubatorfor 6 hours. GFP expression level in the cells was evaluated by flowcytometer (FACS Calibur, BD Bioscience Co. Ltd). The percentage of GFPpositive cells was described in the figure. (FIG. 1B) CAL-1/NFκB-GFPcells (1×10⁵/well) were pre-incubated with (CCT)7, (CCT)8 and (CCT)9(0.1 μM, 0.3 μM, 1.0 μM) for 2 hours. The cells were stimulated withCpG2395 (1 μM) for 6 hours. GFP expression level in the cells wasevaluated by flow cytometer (FACS Calibur, BD Bioscience Co. Ltd). Thepercentage of GFP positive cells in each condition was described in thefigure.

<Experimental Result>

As shown in FIG. 1A, GFP was induced in CAL-1/NFκB-GFP cells by CpG2395stimulation, indicating that activation of NF-κB was induced by TLR9stimulation. Further, this GFP expression was blocked by the addition ofinhibitory-ODNs. As higher concentration of inhibitory-ODNs showedbetter inhibition for the induction of GFP expression in CAL-1/NFκB-GFPcells, dose dependency of inhibitory activity was confirmed (maximuminhibition was observed at 1.0 μM of each inhibitory-ODN). (CCT)9blocked GFP expression with better efficacy than that of (CCT)8 or(CCT)7. These data indicate that inhibitory-ODNs we examined can preventNF-κB activation induced by TLR9 agonist in human cell line system.

Example 2

Comparison of Suppression Activity of Inhibitory-ODNs on NF-κBActivation Induced by TLR9 Stimulation

<Experimental Method>

CAL-1/NFκB-GFP cells (1×10⁵/well) were pre-incubated with variousinhibitory-ODNs described above for 2 hours. The cells were stimulatedwith CpG2395 (1 μM) for 6 hours. GFP expression level of the cells ineach condition was evaluated by flow cytometer (FACS Calibur, BDBioscience Co. Ltd). The percentage of GFP positive cells with CpG2395alone was defined as 100% in the graph. The percentage of GFP positivein each condition was calculated from the number.

<Experimental Result>

As shown in FIG. 2A, dose dependency of the inhibitory activity forNF-κB activation was confirmed in each inhibitory-ODN. (CCT)8 inhibitedGFP expression induced by CpG2395 and showed better activity than (CCT)6and (CCT)7 in human pDC cell line. (CCT)9 strongly blocked GFPexpression with better efficacy than (CCT)8. (CCT)10, (CCT)11 and(CCT)12 exhibited much better inhibitory activity than (CCT)9. Theseresults suggest that longer ODNs have better activity than shorter ODNs.However, The inhibitory activity of (CCT)14 and (CCT)16 was as same asthe activity of (CCT)12 (FIG. 2B), suggesting that (CCT)12 as well as(CCT)14 and (CCT)16 may have maximum efficacy for the inhibition ofNF-κB activity, which induced by TLR9 stimulation. Importantly, theinhibitory activity of (CCT)8 at 1.0 μM was almost same as those of(CCT)11 and (CCT)12 at 0.1 μM. This data indicate that (CCT)11 and(CCT)12 have ten times higher efficacy for the inhibition of NF-κBactivation than (CCT)8 in human cells. As shown in FIGS. 2C and 2D,(CCT)8C and (CCT)8CC exhibited better inhibitory activity than (CCT)8.It was also demonstrated that (CCT)10C and (CCT)10CC exhibited betterinhibitory activity than (CCT)10. Further, (CCT)11C and (CCT)11CC hadbetter inhibitory activity than (CCT)11, while the inhibitory activityof (CCT)11 was already almost saturated. These results indicated thatthe addition of C or CC at the 3′ end of (CCT) repeats increased theinhibitory activity of the ODNs.

It is well established activated NF-κB further induce the production ofproinflammatory cytokines such as interleukin-6 (IL-6) and tumornecrosis factor alpha (TNFα). As oligonuleotides (ODNs) we examinedstrongly inhibits NF-κB activation induced by TLR stimulation, the ODNscan be used as a remedy for the treatment of diseases related to NF-κBactivation. As NF-κB activation has been reported to contribute to thedevelopment of autoimmune diseases such as rheumatoid arthritis,gastritis and inflammatory bowel disease, the ODNs we examined can beused as a remedy for the treatment of the diseases by inhibiting theNF-κB activation.

Example 3

Comparison of Suppression Activity of Inhibitory-ODNs on ProinflammatoryCytokines Production Induced by TLR9 Stimulation for Human Cells

<Experimental Method>

Human plasmacytoid DC cell line; CAL-1 cells were cultured (1×10⁵/well)were plated in 96-well flat-bottomed plate (Costar) and stimulated withCpG2395 (0.4 μM) in the presence of inhibitory-ODNs for 24 hours(concentrations of the inhibitory-ODNs are described in the figure).After 24 hours stimulation, cultured supernatants were recovered andproinflammatory cytokines production was evaluated. The level of IL-6and TNFα production were measured by ELISA as described in manufacture'sprotocol (R&D systems Co. Ltd, Minneapolis, USA).

<Experimental Result>

As shown in FIG. 3, both IL-6 and TNFα production induced by CpG2395 wasblocked by the addition of inhibitory-ODNs in CAL-1 cells. Dosedependency of the inhibitory activity for IL-6 and TNFα production wasconfirmed in each inhibitory-ODN. (CCT)9, (CCT)10, (CCT)11 and (CCT)12strongly blocked both IL-6 and TNFα production induced by CpG2395. Theefficacy of these ODNs was much better than that of (CCT)8. Importantly,the inhibitory activity of (CCT)8 at 0.4 μM was almost same as those of(CCT)11 and (CCT)12 at 0.04 μM. This data indicates that (CCT)11 and(CCT)12 have ten times higher efficacy than (CCT)8 in human cells. As(CCT)11 and (CCT)12 exhibited almost 100% inhibition at 0.04 uM, wefurther evaluated the inhibitory activity at lower concentrations (FIG.4). As shown in the figure, (CCT)9 and (CCT)10 blocked TNFα productionwith much better efficacy than (CCT)8. Further, (CCT)11, (CCT)12,(CCT)14 and (CCT)16 exhibited strong efficacy for the inhibition of TNFαproduction at very low dose in human cells. These results suggest thatthe ODNs can be used as a remedy for the treatment of variousimmune-mediated disorders such as autoimmune diseases, graft rejection,hypersensitivity, diseases associated with the over-stimulation by ofhost's immune system by autoantigens and microbes. As it has beenreported that IL-6 and TNFα play key roles for the development of thediseases such as rheumatoid arthritis, gastritis and inflammatory boweldisease, the ODNs we examined can be used a remedy for the treatment ofthe diseases by the inhibition of IL-6 and TNFα.

Example 4

Comparison of Suppression Activity of Inhibitory-ODNs on ProinflammatoryCytokines Production Induced by TLR9 Stimulation for Mouse Cells

<Experimental Method>

Mouse DC cell line; D2SC/1 cells were cultured D2SC/1 (1×10⁵/well) wereplated in 96-well flat-bottomed plate (Costar) and stimulated withCpG1826 (0.65 μM) in the presence of inhibitory-ODNs for 24 hours(concentrations of the inhibitory-ODNs are described in the figure).After 24 hours stimulation, cultured supernatants were recovered andproinflammatory cytokines production was evaluated. The level of IL-6and TNFα production were measured by ELISA as described in manufacture'sprotocol (R&D systems Co. Ltd, Minneapolis, USA).

<Experimental Result>

As shown in FIG. 5A, both IL-6 and TNFα production induced by CpG1826was blocked by the addition of inhibitory-ODNs in mouse DC cell line;D2SC/1 cells. Dose dependency of the inhibitory activity for IL-6 andTNFα production was confirmed in each inhibitory-ODN. (CCT)9, (CCT)10,(CCT)11, (CCT)12, (CCT)14 and (CCT)16 strongly blocked both IL-6 andTNFα production induced by CpG1826. Importantly, the efficacy of theseODNs was much better than that of (CCT)8. As shown in FIG. 5B, (CCT)8 at0.1 μM barely inhibited TNFα production which induced by CpG1826.However, (CCT)10, (CCT)11, (CCT)12, (CCT)14 and (CCT)16 at sameconcentration strongly inhibited TNFα production. This data indicatesthat (CCT)10, (CCT)11, (CCT)12, (CCT)14 and (CCT)16 have much betterinhibitory effects than (CCT)8 for TLR9 stimulation in mouse cells.

It was documented that D-galactosamin presensitized mice with CpG ODNdeveloped cytokine-mediated lethal shock because of the induction ofhyper immune reactions (Peter M, et al. Immunology. 2008 January;123(1):118-28). Analyses of plasma cytokines revealed over-production ofproinflammatory cytokines such as TNFα (Marshall A J, et al. InfectImmun 1998 April; 66(4):1325-33; Peter M, Bode K, et al. Immunology.2008 January; 123(1):118-28). The ODNs we evaluated strongly inhibitsthe production of TNF from mouse cells induced by TLR9 stimulation.Because the cytokine-mediated lethal shock contributes to the septicshock (Slifka M K, et al. J Mol Med. 2000; 78(2):74-80; Espat N J, etal. J Surg Res. 1995 July; 59(1):153-8) and multiple organ dysfunctionsyndromes (MODS) (Wang H, et al. Am J Emerg Med. 2008 July;26(6):711-5), the ODNs we evaluated can be used as a remedy for thetreatment of sepsis and MOGS by rescuing the host from cytokine-mediatedlethal shock.

Example 5

The Suppression Activity of Inhibitory-ODNs on IFNα Production fromHuman PBMCs Stimulated with TLR9 Agonist.

<Experimental Method>

Human peripheral mononuclear cells (huPBMCs), used in the followingsamples, were isolated from peripheral blood by Ficoll-Hypaque(Pharmacia) density gradient centrifugation (P. M. Daftarian et al.,(1996): Journal of Immunology, 157, 12-20). The cells were cultured inRPMI supplemented with 10% FCS (v/v) and antibiotics (100 IU ofpenicillin/ml and 100 IU of streptomycin/ml) at 37° C. in a 5% CO₂humidified incubator. IFNα production from PBMCs induced by TLR9stimulation was evaluated. Briefly, huPBMCs (5×10⁶/ml) were plated into96 well flat-bottomed plate and stimulated with CpG2216 (1 μM) in thepresence of inhibitory-ODNs (0.1 μM); (CCT)8, (CCT)9, (CCT)10, (CCT)11,(CCT)12, (CCT)14 and (CCT)16. The culture supernatants were collectedfor measuring the level of IFNα production. The level of IFNα productionwas measured by ELISA as described in manufacture's protocol (R&Dsystems Co. Ltd, Minneapolis, USA).

<Experimental Results>

As shown in FIG. 6, huPBMCs produced IFNα in response to TLR9 agonist;CpG2216. (CCT)8 blocked IFNα production induced by CpG2216. However, thesuppression efficacy of (CCT)8 was not so strong. (CCT)9, (CCT)10,(CCT)11, (CCT)12, (CCT)14 and (CCT)16 exhibited better inhibitoryactivity for IFNα production by CpG2216 than (CCT)8. Especially,(CCT)11, (CCT)12, (CCT)14 and (CCT)16 strongly inhibited IFNα productioninduced by CpG2216. These results indicate that the inhibitory-ODNs weevaluated can be inhibitor of TLR9 and IFNα production in human PBMCs.It is well established that elevated production of IFNs contributes tothe development of SLE (Barrat F J, et al. J Exp Med 2005; 202:1131-9;Wellmann U, et al. Proc Natl Acad Sci USA 2005; 102:9258-63). It hasbeen demonstrated that endogenous IFN inducing factors has been reportedexisted in the serum of SLE patient (Kwok S K, et al. Arthritis ResTher. 2008; 10(2):R29), SLE patients have a circulating inducer of IFNproduction, sera from SLE patient frequently induce the production ofIFN via TLR9 in cultures of PBMC from healthy blood donors. As the ODNswe examined could efficiently block IFNα production, the ODNs weevaluated can be used as a remedy for the treatment of SLE patients byinhibiting IFN production.

Example 6

Comparison of Suppression Activity of Inhibitory-ODNs on NF-κBActivation Induced by TLR7/8 Stimulation

<Experimental Method>

CAL-1/NFκB-GFP cells (1×10⁵/well) were pre-incubated for 2 hours withinhibitory-ODNs described previously. The cells were stimulated withTLR7/8 agonist; Gardiquimod or CL264 (Invivogen, USA), for 4 hours. GFPexpression level of the cells in each condition was evaluated by flowcytometer (FACS Calibur, BD Bioscience Co. Ltd). FIG. 7 (A)CAL-1/NFκB-GFP cells were stimulated with TLR7/8 agonist; Gardiquimod (2μg/ml) for 4 hours in the presence of (CCT)6, (CCT)7 and (CCT)8 (0.1 uM,0.3 uM and 1.0 uM). The percentage of GFP positive cells withGardiquimod alone was defined as 100% in the graph. The percentage ofGFP positive in each condition was calculated from the number. FIG. 7(B) CAL-1/NFκB-GFP cells were stimulated with TLR7/8 agonist; CL264 (1μg/ml) for 4 hours in the presence of (CCT)8, (CCT)9, (CCT)10, (CCT)11,(CCT)12, (CCT)14 and (CCT)16 (0.01 uM, 0.03 uM and 0.1 uM). Thepercentage of GFP positive in each condition was calculated as describedpreviously.

<Experimental Results>

As shown in FIG. 7A, GFP expression was induced in CAL-1/NFκB-GFP cellsby Gardiquimod stimulation, indicating that activation of NF-κB wasinduced by TLR7 stimulation. Further, this GFP expression was blocked bythe addition of inhibitory-ODNs. Dose dependency of the inhibitoryactivity for NF-κB activation by TLR7 stimulation was confirmed in eachinhibitory-ODN. (CCT)6 and (CCT)7 showed better activity for Gardiquimodstimulation than (CCT)8, while (CCT)8 also blocked GFP expression.Importantly, the inhibitory activity of (CCT)8 at 1.0 μM was almost sameas those of (CCT)6 and (CCT)7 at 0.1 μM. This data indicates that (CCT)6and (CCT)7 have ten times higher efficacy for the inhibition of NF-κBactivation, which induced by TLR7 stimulation, than (CCT)8. As shownFIG. 2, (CCT)8 showed better inhibitory activity for TLR9 stimulationthan (CCT)6 and (CCT)7. Thus, this suggests (CCT)6 and (CCT)7 haveunique inhibitory activity for TLR7, but not TLR9, stimulation. As shownFIG. 7B, GFP expression was induced in CAL-1/NFκB-GFP cells by CL264stimulation and this GFP expression was blocked by the addition ofinhibitory-ODNs. (CCT)9 (CCT)10, (CCT)11 and (CCT)12 efficiently blockedGFP expression by CL264 stimulation and exhibited better inhibitoryactivity than (CCT)8. These results suggest that longer ODNs have betterinhibitory activity for TLR7 stimulation, however the inhibitoryactivity of (CCT)14 and (CCT)16 for TLR7 stimulation was much worse thanthe activity of (CCT)12. This indicates that (CCT)12 may have maximumefficacy for the inhibition of NF-κB activity, which induced by TLR7stimulation. Our data provide the ODNs we examined can block TLR7stimulation in human cells. It has been demonstrated that uncontrolledIFN production contributes to the development of SLE (Barrat F J, et al.J Exp Med 2005; 202:1131-9; Wellmann U, et al. Proc Natl Acad Sci USA2005; 102:9258-63) and IFN production from huPBMCs was produced by TLR7stimulation. Together with the results of the example, the ODNs weexamined can be used as a remedy for the treatment of TLR-mediateddisease such as SLE by inhibiting TLR7 or TLR9 activation.

Other embodiments are within the following claims. While severalembodiments have been shown and described, various modifications may bemade without departing from the spirit and scope of the presentinvention.

1-26. (canceled)
 27. A method of treating an immune-mediated disorder ina human subject in need thereof, comprising administering to the humansubject an oligonucleotide comprising a sequence selected from the groupconsisting of: (SEQ ID NO: 15) 5′-cctcctcctcctcctcct-3′,  and(SEQ ID NO: 16) 5′-cctcctcctcctcctcctcct-3′.


28. The method of claim 27, wherein the immune-mediated disorder is anautoimmune disease, hypersensitivity, graft rejection, a diseaseassociated with over-stimulation of host's immune system by microbes, anNF-kB mediated disease or a Toll-like receptor (TLR)-mediated disease.29. The method of claim 27, wherein the immune-mediated disorder is aTLR-mediated disease and the oligonucleotide inhibits the proliferationof immune cells.
 30. The method of claim 27, wherein the immune-mediateddisorder is a TLR-mediated disease and the oligonucleotide inhibits theactivation of TLR7 and/or TLR8.
 31. The method of claim 27, wherein theimmune-mediated disorder is a NF-kB-mediated disease and theoligonucleotide inhibits the activation of NF-kB.
 32. The method ofclaim 27, wherein the oligonucleotide inhibits interferon production.33. The method of claim 27, wherein the oligonucleotide inhibitsproinflammatory cytokine production.
 34. The method of claim 27, whereinthe oligonucleotide rescues the subject from cytokine-mediated lethalshock.
 35. The method of claim 27, wherein the immune-mediated disorderis systemic lupus erythematosus (SLE).
 36. The method of claim 27,wherein the immune-mediated disorder is rheumatoid arthritis, gastritis,or inflammatory bowel disease.
 37. The method of claim 27, wherein theimmune-mediated disorder is sepsis or multiple organ dysfunctionsyndrome.
 38. The method of claim 27, wherein the oligonucleotide isadministered alone or with a pharmaceutically acceptable carrier to asubject having or at risk of developing the immune-mediated disorder.39. The method of claim 27, wherein the oligonucleotide is administeredto the subject via enteral, parenteral and topical administration orinhalation.
 40. The method of claim 27, wherein the oligonucleotide isadministered to the subject in a pharmaceutical composition.
 41. Themethod of claim 27, wherein the oligonucleotide is pegylated.
 42. Themethod of claim 27, wherein the phosphate backbone of theoligonucleotide is partly or completely phosphorothioate-modified. 43.The method of claim 27, wherein the phosphate backbone of theoligonucleotide is unmodified.
 44. The method of claim 27, wherein theoligonucleotide consists of the sequence selected from the groupconsisting of (SEQ ID NO: 15) 5′-cctcctcctcctcctcct-3′,  and(SEQ ID NO: 16) 5′-cctcctcctcctcctcctcct-3′.


45. A method of treating an immune-mediated disorder in a human subjectin need thereof, comprising administering to the human subject anoligonucleotide comprising a sequence selected from the group consistingof: (SEQ ID NO: 15) 5′-cctcctcctcctcctcct-3′,  and (SEQ ID NO: 16)5′-cctcctcctcctcctcctcct-3′,

wherein a nucleotide is added to, deleted from, or added to and deletedfrom either end of the sequence.
 46. The method of claim 45, wherein theoligonucleotide consists of the sequence selected from the groupconsisting of (SEQ ID NO: 15) 5′-cctcctcctcctcctcct-3′,  and(SEQ ID NO: 16) 5′-cctcctcctcctcctcctcct-3′,

wherein a nucleotide is added to, deleted from, or added to and deletedfrom either end of the sequence.